Anti-clogging device and method for in-gel digestion applications

ABSTRACT

An integrated proteomics sample preparation device and method for in-gel digestion of proteins and for desalting and concentrating samples prior to further analysis such as by MALDI TOF and/or electro-spray ionization (ESI) mass spectrometry. The device and method of the present invention allow for digestion, desalting and concentration of sample prior to analysis. More specifically, the device in accordance with an embodiment of the present invention includes a plurality of wells in fluid communication with a an outlet or drainage opening containing a three dimensional structure comprising a plurality of sorptive particles entrapped in a porous polymer matrix so as to form a device capable of carrying out solid phase extraction. In a preferred embodiment, the wells are configured so as to prevent a sample carrier present in the wells from clogging the outlet when subjected to a driving force such as vacuum. The device also reduces or eliminates overflowing of a well in the event a drain becomes clogged during automated operation.

This application is a continuation of U.S. patent application Ser. No.10/154,550 filed May 24, 2002, the disclosure of which is incorporatedherein by reference

BACKGROUND OF THE INVENTION

Matrix-assisted laser desorption/ionization (MALDI) analysis is a usefultool for solving structural problems in biochemistry, immunology,genetics and biology. Samples are ionized and a time of flight (TOF)analyzer is used to measure ion masses. TOF analysis begins when ionsare formed and are accelerated to a constant kinetic energy as theyenter a drift region. They arrive at a detector following flight timesthat are proportional to the square root of their masses. A massspectrum is created because ions of different mass arrive at thedetector at different times.

Mass spectrometry can be a particularly powerful tool in the fields ofdrug discovery and development, genotyping, and proteome research.Current trends in research are to analyze larger and larger numbers ofsamples using automated handling equipment or robotics. Quantities ofindividual samples are from the nano-mole levels to femto-mole levels.As a result, instrumentation is becoming more sensitive and a needexists for sample handling formats to be miniaturized, high density anddisposable.

In-gel digestion of protein is a proteomics method that has many samplepreparation steps prior to sample analysis (such as by MALDI TOF MS).Briefly, upon separation in the electrophoresis gel, the proteins in asample are stained for detection and portions of the gel containing theprotein of interest are excised. The stain is then removed from thesegel portions, and an enzyme solution is used to selectively digest theprotein sample to form peptides that migrate out of the gel portion intosolution. After purification of the peptides, analysis of the sample iscarried out.

Simultaneous preparation and analysis of multiple samples is oftendesirable. Multiwell plates have been developed for simultaneous assay,typically consisting of 96, 384 or 1536 reaction vessels or wells perplate. It would be desirable to use multiwell plates also for samplehandling and preparation, such as the removal of undesired salts andbiochemical substances to improve the resolution and selectivity of themass spectrum.

In this connection, EP 1 151 793 discloses a microtiter plate havinglyophobic porous bottoms. Gel pieces containing proteins are placed inthe wells of the plate and digested with enzyme. The enzyme is thenremoved from the gel pieces by centrifugation and applied to a MALDIsample carrier plate for analysis.

However, using centrifugation to bind, wash and elute is atime-consuming process. In addition, it is not easily adaptable toautomation or robotics. It would be highly desirable to use themicrotiter plate format for enzyme digestion and protein capture thatdoes not require centrifugation, and that is readily adaptable toautomation.

Another difficulty is that the gel plugs are deformable and have asimilar diameter to the cone-shaped drain outlet of the plate. Whenvacuum filtered, the gel plugs clog the outlet, causing the well eitherto not drain or overflow with multiple solution additions, thuscontaminating adjacent wells.

It is therefore an object of the present invention to provide a samplepreparation method for desalting and purification of samples prior tomatrix assisted laser desorption ionization time-of-flight (MALDI TOF)or electro-spray ionization (ESI) mass spectrometry or other analysismethods, that also can be used for digestion of protein, particularlyin-gel digestion.

It is a still further object of the present invention to provide ahigh-density multi-well device wherein various arrays within the devicecontain chromatographic media having the same or different chemistries,and wherein in-gel digestion of protein is carried out using vacuum as adriving force.

It is a further object of the present invention to provide a samplepreparation system and method that is suitable for automated roboticsliquid handling equipment.

These and other objects will be made apparent by the followingdescription.

SUMMARY OF THE INVENTION

The problems of the prior art have been overcome by the presentinvention, one embodiment of which provides an integrated proteomicssample preparation device and method for digestion of proteins and fordesalting and concentrating samples prior to further analysis such as byMALDI TOF and/or electro-spray ionization (ESI) mass spectrometry. Thedevice and method of the present invention allows for digestion,desalting and concentration of sample prior to MALDI TOF MS analysis.More specifically, the device in accordance with an embodiment of thepresent invention includes a plurality of wells each in fluidcommunication with a respective outlet or drainage opening, optionallycontaining a three dimensional structure comprising a plurality ofsorptive particles entrapped in a porous polymer matrix so as to form adevice capable of carrying out solid phase extraction. In a preferredembodiment, the wells are configured so as to prevent a sample carrier,such as a gel piece inserted in the wells from clogging the outlet whensubjected to a driving force such as vacuum. The device also reduces oreliminates cross-contamination between wells in the event a drainbecomes clogged.

The present invention is also directed towards a method of samplepreparation using the device of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a single well for a multiwell samplepreparation device in accordance with the present invention;

FIG. 2 is a perspective view of a single well for a multiwell samplepreparation device shown containing a gel piece in accordance with thepresent invention;

FIG. 3 is a cross-sectional view of a single well for a multiwell samplepreparation device shown containing a gel piece in accordance with thepresent invention;

FIG. 4 is a enlarged perspective view of a single well for a multiwellsample preparation device shown containing a gel piece in accordancewith the present invention;

FIG. 5 is a cross-sectional view of a single well for a multiwell samplepreparation device shown containing a gel piece (in phantom) and amatrix having adsorptive properties in the drain in accordance with thepresent invention;

FIG. 5A is a top view of fluid passageways formed in the well inaccordance with the present invention;

FIG. 6 is a perspective view of two side-by-side wells of a multi-wellsample preparation device in accordance with the present invention;

FIG. 7 is a perspective view of a solid rendering of the drain andpassageways of a well in accordance with the present invention;

FIG. 8 is a cross-sectional view of a single well for a multiwell samplepreparation device shown containing a gel piece (in phantom) and amatrix having adsorptive properties in the drain in accordance with analternative embodiment of the present invention;

FIG. 8A is a top view of fluid passageways formed in the well inaccordance with the embodiment of FIG. 8;

FIG. 9 is a perspective view of a well having a dividing member inaccordance with an embodiment of the present invention; and

FIG. 10 is a cross-sectional view of a well having raised bumps inaccordance with an embodiment of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Suitable substrate materials for the sample preparation device of thepresent invention are not particularly limited, and include plastics(such as polyethylene and polypropylene), glass and stainless steel. Thesubstrate materials should not interfere with the operation of thedevice or the chemicals to be used in the procedure. Polyolefins, andparticularly polypropylene, are preferred materials.

Turning now to FIGS. 1 and 2, there is shown generally at 10 a singlewell 12 suitable for use in a single well or a multiwell samplepreparation device that has a plurality of wells. A well 12 is definedby a vertically extending fluid impervious side wall and a slopingbottom portion. The middle and upper portions of the well 12 preferablyhave a uniform diameter and are substantially cylindrical incross-section, although other configurations are contemplated and withinthe scope of the present invention. The lower portion of the well 12tapers downwardly, in the direction of fluid flow, towards a bottomportion 13, which slopes inwardly towards a center, thereby having afrusto-conical configuration. Bottom portion 13 has a drain 15 that ispreferably centrally located in the well 10.

Formed in the bottom portion 13 of the well 10 are one or more fluidpassageways 18. The fluid passageway(s) 18 modify the otherwiserelatively smooth or even surface of bottom portion 13 and effectivelyprovide a gap or space between a sample carrier 20, such as a gel piece(FIG. 2), that is contained in well 12 and supported by the bottomportion 13, and the drain 15. The sample carrier can be a liquid but ispreferably a solid, such as a gel, coated bead or a membrane. In orderto insure fluid flow between the well 12 and drain 15 when the carrier20 is present in the well 12, the smallest dimension of each passageway18 should be less than the smallest dimension of the carrier 20, so thatthe carrier 20 cannot be positioned in the passageway 18 to block fluidflow into the drain 15. In this way, at least a portion of the fluidpassageway(s) 18 is always in fluid communication with the drain 15 andcannot be blocked or clogged by a carrier 20 when placed in the well 12,as exemplified by illustration in FIGS. 2, 3 and 4. Where the carrier isa gel piece, it is noted that typically circular plug cutters found inautomated picker robots cut the gel portion uniformly. However, thepresent invention is not limited to uniformly-shaped carriers, as thefluid passageway(s) 18 are configured to prevent fluid blockage evenwhen carriers of irregular shape are present in the well 12. Forexample, a single slit that is longer than the carrier is within thescope of the present invention.

Although a single passageway 18 is sufficient to insure fluid flowaround the sample carrier, preferably there is a plurality of suchpassageways. At least two passageways 18, most preferably threepassageways 18, formed symmetrically about the drain 15 as best seen inFIGS. 5A and 7, is the particularly preferred arrangement. Thesymmetrical arrangement of the passageways about the drain 15 ensurethat regardless of the orientation of the carrier 20 in the well 12,fluid communication between the well 12 and the drain 15 will bemaintained. The shape and topology of the passageway(s) 18 are notparticularly limited, as long as they do not match that of the carrier20. Preferably the passageway(s) 18 are lobes, but a square, steppedround, cone with a bump or cross bar also are suitable configurations.As best seen in FIG. 7, the lobes preferably taper so that they aredeeper as they approach the drain 15.

The passageway(s) 18 are preferably formed by creating asymmetry in thesurface of the bottom portion 13. This can be accomplished by providinggrooves in the surface, or by providing raised portions or protrusionsin or on the surface such as a cross bar 117 (FIG. 9) or ribs or bumps118 (FIG. 10). Preferably the passageways 18 are grooves having a depthof about 0.2 mm, a width of about 0.25 mm and a length of about 1 mm. Inthe embodiment utilizing protrusions, the protrusions are designed sothat the largest opening in the drain is smaller than the smallestdimension of the sample carrier. The objective is to prevent the samplecarrier 20 from being situated over the drain 15 in such as way as toblock fluid flow to the drain 15.

As seen in FIGS. 1 and 7, the drain 15 is a bore, preferably cylindricaland axially aligned with the central longitudinal axis of the well 12.The drain 15 is in fluid communication with the passageways 18. At leasta portion of the drain 15 preferably includes an adsorptive compositestructure 25 (FIGS. 5 and 5A). Suitable adsorptive composite structuresare cast-in-place polymer bound, particle laden adsorptive membranestructures, such as those comprised of chromatographic beads which havebeen adhered together with a binder and disclosed in U.S. Pat. No.6,048,457, the disclosure of which is hereby incorporated by reference.One such preferred structure is a three-dimensional structure comprisinga plurality of sorptive particles entrapped in a porous polymer matrixand having an aspect ratio (average diameter to average thickness) ofless than about 10, preferably less than about 5. The structure 25 ispreferably coterminous with the bottom of the drain 15, and extends intothe drain 15, preferably extending through the entire depth of the drain15 and may extend into the passageway(s) 18 as shown in FIG. 5. Althoughthe composite structure 25 can also completely fill the passageway(s)18, it is preferred that a portion (preferably the upper half), such as50%, of the passageway(s) 18 remains devoid of structure 25 to ensurethe passageway(s) 18 is not blocked by the carrier 20.

As shown in FIGS. 8 and 8A, the composite structure can be formed tohave one or more dimensions that are greater than the largest dimensionof the carrier 20, and thus ensure fluid communication between the welland the drain without the formation of a passageway to maintain surfacearea for flow. For example, the face shape of the composite structurecan be a circle having a long leg 25A extending from the circle, or canbe in the shape of an eye, thereby ensuring that some surface of thecomposite structure remains unobstructed and available for flowregardless of the orientation of the carrier 20.

Devices in accordance with the present invention may incorporate aplurality of composite structures having resin materials with differentfunctional groups to fractionate analytes that vary by charge, size,affinity and/or hydrophobicity; alternately, a plurality of devicescontaining different individual functional membranes may be used incombination to achieve a similar result. Similarly, one or moremembranes can be cast in a suitable housing and functionality can beadded before or after casting.

In an alternative embodiment, the drain can be devoid of any media, andthe device used as a non-clogging processing device that deliversdigested proteins to a collection well for analysis or concentration,for example.

After the proteins in the carrier are stained and small pieces of thecarrier containing the protein(s) of interest are excised from the siteof the stain, each carrier piece is placed in a respective well. Asuitable amount of proteolytic enzyme solution is added to each well,such as by pipetting. Sufficient enzyme is added to effectively digestthe protein(s). Preferably an excess of enzyme is added, and insufficient amount to submerge the carrier in each well. After anincubation period to allow the protein digestion to take place and theresulting peptides to diffuse out of the carrier, vacuum is applied toeach well, preferably to create a pressure differential of about 5-10psi, to cause extracted peptides to flow into the drain 15 where theyare adsorbed (when media is present) and can then be washed in theconventional manner and freed from buffers, salts and othercontaminants. Concentrated peptides then can be eluted and delivered toa suitable target or presentation device for analysis such as by MALDITOF MS.

During an automated multi-addition procedure, there is the possibilitythat wells can overflow, if blocked. The present invention reduces oreliminates the possibility of contamination of other wells as a resultof the overflow by incorporating an overflow control feature into thedevice of the present invention. Specifically, with reference to FIG. 6,surrounding at least a portion of each well 12 is a recess 30. Therecess preferably is formed from the top surface 29 of each well 12,which generally corresponds to the top surface 32 of the tray 35, andextends downward (towards the drain 15) about 50% of the length of thewell 12, where it terminates in bottom wall 34. The depth of the recessis not critical, as long as is sufficient to contain the overflow volumefrom at least one well.

1.-8. (canceled)
 9. A sample preparation assembly, comprising: a samplecarrier, a sample preparation device in which said sample carrier ispositioned, said device comprising at least one well having an inlet, abottom surface, a drain in said bottom surface, and at least one fluidpassageway formed in said bottom surface about said drain; said at leastone fluid passageway having a dimension smaller than the smallestdimension of said carrier such that fluid communication between saidwell inlet and said drain, around said sample carrier, is maintainedwhen said carrier is in said well and is positioned on said at least onefluid passageway, said at least one fluid passageway tapering such thatit is deeper as it approaches said drain; and a source of vacuum influid communication with said well.
 10. The assembly of claim 9, whereinsaid at least one fluid passageway has a dimension less than 1.0 mm. 11.The assembly of claim 9, wherein there are three passageways arrangedsymmetrically about said drain.
 12. The assembly of claim 9, wherein anadsorptive structure is positioned in said drain.
 13. The assembly ofclaim 12, wherein said structure comprises a plurality of sorptiveparticles entrapped in a porous matrix.
 14. The assembly of claim 9,wherein said sample carrier comprises a gel.
 15. The assembly of claim9, wherein there are a plurality of wells.
 16. The assembly of claim 15,wherein said plurality of wells are surrounded by a recess. 17.-21.(canceled)
 22. A sample preparation assembly, comprising: a samplecarrier, a sample preparation device in which said sample carrier ispositioned, said device comprising at least one well having an inlet, abottom surface, a drain in said bottom surface, and at least one grooveformed in said bottom surface about said drain; said at least one groovehaving a dimension smaller than the smallest dimension of said carriersuch that fluid communication between said well inlet and said drain,around said sample carrier, is maintained when said carrier is in saidwell and is positioned on said at least one groove, said at least onegroove tapering such that it is deeper as it approaches said drain; anda source of vacuum in fluid communication with said well.
 23. Theassembly of claim 22, wherein there are three grooves arrangedsymmetrically about said drain.
 24. The assembly of claim 22, wherein anadsorptive structure is positioned in said drain.
 25. The assembly ofclaim 24, wherein said structure comprises a plurality of sorptiveparticles entrapped in a porous matrix.
 26. The assembly of claim 22,wherein said sample carrier comprises a gel.
 27. The assembly of claim22, wherein there are a plurality of wells.
 28. The assembly of claim27, wherein said plurality of wells are surrounded by a recess.